Hemagglutinin-binding peptide

ABSTRACT

Object of the present invention is to provide a hemagglutinin-binding peptide producing an anti-influenza virus effect higher than that of existing peptides. The present invention provides, for example, a hemagglutinin-binding peptide comprising a polypeptide having any of the following amino acid sequences (i) to (iv):
         (i) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Arg (SEQ ID NO: 1);   (ii) Arg-Val-Ser-MePhe-Thr-Tyr-MePhe-MeSer-Tyr-Thr-Pro-Ser (SEQ ID NO: 2);   (iii) an amino acid sequence with deletions, additions, or substitutions of one or several amino acids in SEQ ID NO: 1 or 2; and   (iv) an amino acid sequence having 90% or more sequence identity to that of SEQ ID NO: 1 or 2.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The content of the ASCII text file of the sequence listing named“20170421_101620_001US1_seq”, which was filed in PCT/JP2015/079931,downloaded from the WIPO database, is 27.0 kb in size with a createddate of May 10, 2017, and electronically submitted via EFS-Web on Apr.21, 2017, is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This application claims priority from Japanese Patent Application No.2014-217582, filed on Oct. 24, 2014, the details of which areincorporated herein by reference.

The present invention relates to a hemagglutinin-binding peptide havinganti-influenza virus activity, and the like.

BACKGROUND ART

Emergence and spread of a new influenza virus have historically causedserious damage to humankind. Even now, a highly pathogenic avianinfluenza virus is expected to pose large threats when it mutates into avirus capable of human-to-human transmission.

Influenza virus is an enveloped RNA virus and it is categorized intotypes A, B, and C according to the antigenicity of nucleoprotein (NP)and matrix protein (M). Type A and type B are most common epidemic inhuman beings.

Even if viruses belong to the same type such as type A, B, or C, theyare sub-divided into different serotypes such as H1N1, H5N1, or the likebased on difference in antigenicity of hemagglutinin (HA) orneuraminidase (NA), each a molecule on the surface of the envelope.

As an existing anti-influenza drug, zanamivir (Relenza™) (for example,Patent Document 1) and oseltamivir (Tamiflu™) (for example, PatentDocument 2) have been used widely.

Zanamivir or oseltamivir suppresses activity of neuraminidase whichbecomes necessary for infection of an influenza virus from infectedcells to other cells. Anti-influenza drugs having another workingmechanism include amantadine and rimantadine targeting the matrixprotein 2 (M2 protein). They interfere with uncoating of the virus inthe infected cells.

Similar to other drugs targeting an enzyme or ion channel, these drugsthat inhibit the function of neuraminidase or M2 protein have beendeveloped through derivatization from a substrate, molecular designingbased on a steric structure, or discovery of a function from existingcompounds. They therefore belong to an analog of a molecule present in aliving body or a chemically prepared low molecule.

M2 protein Inhibitors are however effective only for influenza type Aand neuraminidase inhibitors do not have efficacy againstneuraminidase-free influenza type C.

Anti-viral drugs generally have a limit in its efficacy against virusesthat have mutated and therefore acquired resistance to the drugs.Emergence of viruses resistant to popularly used anti-influenza virusdrugs such as zanamivir, oseltamivir, and amantadine has also beenfound. Viruses acquire resistance to anti-viral drugs because ofdeterioration in affinity of the drugs for a target molecule due tomutation thereof and further, recovery of proliferation potency broughtby mutation of another molecule, which is an indirect mechanism. It istherefore difficult to create a drug capable of completely preventingacquisition of resistance for a long period of time.

The present inventors have already found a peptide exhibiting inhibitoryactivity against influenza viruses by binding to hemagglutinin (PatentDocument 3).

CITATION LIST

Patent Document 1: U.S. Pat. No. 5,360,817

Patent Document 2: U.S. Pat. No. 5,763,483

Patent Document 3: Japanese Patent Laid-Open No. 2013-071904

SUMMARY OF THE INVENTION Technical Problem to be Solved

Anti-influenza virus drugs targeting hemagglutinin have not yet beenused popularly so that such peptide drugs are presumed to have ananti-viral effect against influenza viruses having resistance to aneuraminidase inhibitor or M2 protein inhibitor. There is however ademand for a hemagglutinin-binding peptide having a higheranti-influenza virus effect.

Many of anti-influenza virus drugs so far used are not effective unlessadministered to the initial stage of infection so that there is a demandfor a medicament effective when administered even several days afterinfection.

With such situations as a background, an object of the present inventionis to provide a hemagglutinin-binding peptide having a highanti-influenza virus effect.

Solution to Solve the Problem

Proceeding with intensive investigation with a view to solving theabove-described problem, the present inventors have found peptideshaving a markedly high anti-influenza virus effect. They have confirmedthat some of these peptides have a sufficient anti-influenza viruseffect even administered after the elapse of a certain time afterinfection, leading to completion of the present invention.

The present invention provides:

[1] a hemagglutinin-binding peptide comprising a polypeptide having anyof the following amino acid sequences (i) to (iv):

(i)  (SEQ ID NO: 1) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Arg, (ii)  (SEQ ID NO: 2)Arg-Val-Ser-MePhe-Thr-Tyr-MePhe-MeSer-Tyr-Thr-Pro- Ser;

(iii) an amino acid sequence with deletions, additions, or substitutionsof one or several amino acids in SEQ ID NO: 1 or 2; and

(iv) an amino acid sequence having 90% or more sequence identity to thatof SEQ ID NO: 1 or 2;

[2] the hemagglutinin-binding peptide described above in [1], comprisinga polypeptide having any of the following amino acid sequences (v) to(vii):

(v) an amino acid sequence with deletions, additions, or substitutionsof one or several amino acids at a position selected from positions 3,6, 12, and 13 in SEQ ID NO: 1,

(vi) an amino acid sequence having substitutions of one or several aminoacids at a position selected from positions 3, 6, and 13 in SEQ ID NO:1, and

(vii) an amino acid sequence having a substitution of an amino acid atposition 13 in SEQ ID NO: 1;

[3] a hemagglutinin-binding peptide containing a polypeptide having anyof the following amino acid sequences (viii) to (xviii):

(viii)  (SEQ ID O: 36) Thr-MeGly-Lys-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Arg; (ix)  (SEQ ID NO: 37)Thr-MeGly-Asp-MePhe-MePhe-Ala-MeSer-His-Tyr-Thr- Val-Pro-Arg; (x) (SEQ ID NO: 38) Thr-MeGly-Asp-MePhe-MePhe-Lys-MeSer-His-Tyr-Thr-Val-Pro-Arg; (xi)  (SEQ ID NO: 39)Thr-MeGly-Asp-MePhe-MePhe-Glu-MeSer-His-Tyr-Thr- Val-Pro-Arg; (xii) (SEQ ID NO: 40) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Hyp-Arg; (xiii)  (SEQ ID NO: 41)Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr- Val-Pro-Ala; (xiv) (SEQ ID NO: 42) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Glu; (xv)  (SEQ ID NO: 43)Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr- Val-Pro-Lys; (xvi) (SEQ ID NO: 44) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Dap;

(xvii) an amino acid sequence with deletions, additions, orsubstitutions of one or several amino acids in any of SEQ ID NOs: 36 to44; and

(xviii) an amino acid sequence having 90% or more sequence identity tothat of any of SEQ ID NOs: 36 to 44;

[4] a hemagglutinin-binding peptide containing an amino acid sequencerepresented by the following formula (I):

(i) (SEQ ID NO: 3) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃ (I)

(wherein,

Xaa₁ is Ser or Thr,

Xaa₂ is an N-methylamino acid,

Xaa₃ is an arbitrary amino acid,

Xaa₄ is a basic amino acid,

Xaa₅ is Val,

Xaa₆ is a basic amino acid,

Xaa₇ is Tyr,

Xaa₈ is Ser or Thr,

Xaa₉ is Val,

Xaa₁₀ is MePhe,

Xaa₁₁ is Asn,

Xaa₁₂ is MeAla, and

Xaa₁₃ is Val or Ser);

[5] the hemagglutinin-binding peptide described above in [4],

wherein Xaa₂ is MePhe or MeGly,

Xaa₃ is MeGly or Thr,

Xaa₄ is His, and

Xaa₆ is His or Arg;

[6] the hemagglutinin-binding peptide described above in [4] or [5],

wherein saidXaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃ is

Ser-MePhe-MeGly-His-Val-His-Tyr-Ser-Val-MePhe-Asn-MeAla-Val or

Thr-MeGly-Thr-His-Val-Arg-Tyr-Thr-Val-MePhe-Asn-MeAla-Ser;

[7] the hemagglutinin-binding peptide described above in any one of [1]to [6] which is cyclized;

[8] the hemagglutinin-binding peptide described above in [7], having achloroacetylated amino acid within 3 amino acids from the N-terminus andcysteine within 3 amino acids from the C-terminus;

[9] the hemagglutinin-binding peptide described above in [7], havingchloroacetyl-Trp at the N-terminus and Cys at the C-terminus and havingbeen cyclized via a thioether bond therebetween;

[10-1] a pharmaceutical composition for the prevention or treatment ofinfluenza, including the hemagglutinin-binding peptide described abovein any one of [1] to [9];

[10-2] a method of preventing or treating influenza, includingadministering an effective amount of the hemagglutinin-binding peptidedescribed above in any one of [1] to [9];

[11] an influenza virus detection agent, comprising thehemagglutinin-binding peptide described above in any one of [1] to [9];and

[12] an influenza virus detection kit, comprising the influenzadetection agent described above in [11].

Advantageous Effects of Invention

The peptide of the present invention binds to hemagglutinin and therebyexhibits high inhibition activity against influenza viruses so that ithas an anti-viral effect also against influenza viruses havingresistance to a neuraminidase inhibitor or M2 protein inhibitor.

It is presumed that the emergence probability of a drug-resistant mutantcan be lowered by using the peptide of the present invention and a drugtargeting neuraminidase, M2 protein, or the like in combination.

Further, the peptide of the present invention produces a sufficientanti-influenza virus effect even when administered after the elapse of acertain period of time after infection so that it is highly useful as aremedy for influenza.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a picture of plates showing the results of a growth inhibitiontest of the peptides of the present invention against influenza virusH5N1-Vac3 (Example 2);

FIG. 2 is a picture of plates showing the results of a growth inhibitiontest of the peptides of the present invention against highly pathogenicavian influenza viruses A/ws/Hokkaido/1/08 (clade 2.3),A/ws/Mongolia/3/05 (clade 2.2), and A/Vietnam/UT3040/04 (clade 1)(Example 2);

FIG. 3 is a picture of plates showing the results of a growth inhibitiontest of the peptides of the present invention against influenza virusH1N1-pdm2619 (Example 2);

FIG. 4 is a picture of plates showing the results of a growth inhibitiontest of the peptides of the present invention against influenza virusH2N2-Adachi (Example 2);

FIG. 5 is an outline of a test for determining a treatment effect ofcyclic peptide iHA-100 and Zanamivir (Example 3);

FIG. 6 shows a treatment effect of the peptide of the present inventionagainst highly pathogenic avian influenza virus H5N1 A/whooperswan/Hokkaido/1/08 (5×MLD50) (Example 3);

FIG. 7 shows a treatment effect of Zanamivir against highly pathogenicavian influenza virus H5N1 A/whooper swan/Hokkaido/1/08 (5×MLD50)(Example 3); and

FIG. 8 is a picture of plates showing the results of a growth inhibitiontest of the peptides of the present invention against influenza virusH5N1-Vac3 (Example 5).

DESCRIPTION OF EMBODIMENTS

(Peptide)

In one aspect, the hemagglutinin-binding peptide of the presentinvention comprises a polypeptide having any of the following amino acidsequences (i) to (iv):

(i)  (SEQ ID NO: 1) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Arg; (ii)  (SEQ ID NO: 2)Arg-Val-Ser-MePhe-Thr-Tyr-MePhe-MeSer-Tyr-Thr- Pro-Ser;

(iii) an amino acid sequence with deletions, additions, or substitutionsof one or several amino acids in SEQ ID NO: 1 or 2; and

(iv) an amino acid sequence having 90% or more sequence identity to thatof SEQ ID NO: 1 or 2.

In one aspect, the hemagglutinin-binding peptide of the presentinvention further comprises a polypeptide having any of the followingamino acid sequences (v) to (xviii):

(v) an amino acid sequence with deletions, additions, or substitutionsof one or several amino acids at a position selected from positions 3,6, 12 and 13 in SEQ ID NO: 1,

(vi) an amino acid sequence having substitutions of one or several aminoacids at a position selected from positions 3, 6 and 13 in SEQ ID NO: 1,and

(vii) an amino acid sequence having a substitution of an amino acid atposition 13 in SEQ ID NO: 1;

(viii)  (SEQ ID NO: 36) Thr-MeGly-Lys-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Arg (ix)  (SEQ ID NO: 37)Thr-MeGly-Asp-MePhe-MePhe-Ala-MeSer-His-Tyr-Thr- Val-Pro-Arg; (x) (SEQ ID NO: 38) Thr-MeGly-Asp-MePhe-MePhe-Lys-MeSer-His-Tyr-Thr-Val-Pro-Arg; (xi)  (SEQ ID NO: 39)Thr-MeGly-Asp-MePhe-MePhe-Glu-MeSer-His-Tyr-Thr- Val-Pro-Arg; (xii) (SEQ ID NO: 40) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Hyp-Arg; (xiii)  (SEQ ID NO: 41)Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr- Val-Pro-Ala; (xiv) (SEQ ID NO: 42) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Glu; (xv)  (SEQ ID NO: 43)Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr- Val-Pro-Lys; (xvi) (SEQ ID NO: 44) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Dap;

(xvii) an amino acid sequence with deletions, additions, orsubstitutions of one or several amino acids in any of SEQ ID NOs: 36 to44; and

(xviii) an amino acid sequence having 90% or more sequence identity tothat of any of SEQ ID NOs: 36 to 44;

In the above formula, MeGly represents N-methylglycine, MePhe representsN-methylphenylalanine, MeSer represents N-methylserine, and Daprepresents 2,3-diaminopropionic acid.

In one aspect, from the viewpoint of high neutralization activity andinhibition activity against proliferation of influenza viruses, thehemagglutinin-binding peptide of the present invention preferablycomprises a polypeptide having an amino acid sequence with deletions,additions, or substitutions of one or several amino acids in SEQ ID NO:41, 42 or 44 or an amino acid sequence having 90% or more sequenceidentity to any of SEQ ID NO: 41, 42, or 44, with the polypeptide havingan amino acid sequence of SEQ ID NO: 41, 42, or 44 being more preferred.

In the present specification, the term “polypeptide” means two or moreamino acids joined by a peptide bond, for example, 8 to 30 amino acidsjoined by peptide bonds. It may be either linear or cyclic.

In the present specification, the term “amino acid” is used in itsbroadest meaning and it embraces not only natural amino acids but alsoderivatives thereof and artificial amino acids. The derivatives eveninclude, for example, amino acids obtained by modifying a natural aminoacid constituting a protein. Examples of such amino acids includehydroxyproline and hydroxylysine, which are amino acids having ahydroxyl group introduced therein, and diaminopropionic acid, which isan amino acid having an amino group introduced therein.

In the present specification, examples of the amino acid include naturalprotein L-amino acids, unnatural amino acids, and chemically synthesizedcompounds having properties known in the art as characteristics of anamino acid. Examples of the unnatural amino acids include, but notlimited to, α,α-disubstituted amino acids (such as α-methylalanine),N-alkyl-α-amino acids, D-amino acids, β-amino acids, and α-hydroxyacids, each having a backbone structure different from that of naturalamino acids; amino acids (such as norleucine and homohistidine) having aside-chain structure different from that of natural amino acids; aminoacids (such as “homo” amino acids, homophenylalanine, and homohistidine)having extra methylene in the side chain thereof; and amino acids (suchas cysteic acid) obtained by substituting a carboxylic acid functionalamino group in the side chain thereof by a sulfonic acid group.

In the present specification, amino acids other than 20 natural aminoacids to be used for expression on the ribosome in living cells, thatis, amino acids corresponding to the following (1) to (3) may be called“non-canonical amino acids”.

(1) Amino acids corresponding to an amino acid residue on a polypeptidesubjected to modification after expression (ex. phosphorylated tyrosine,acetylated lysine, or farnesylated cysteine).

(2) Amino acids that cannot be used in expression on a ribosome butoccur naturally.

(3) Artificial amino acids that do not occur naturally (unnatural aminoacids).

Examples of the non-canonical amino acids are listed in the followingtable, but are not limited to them. In the table, DBE and CME are estersused when a non-canonical amino acid is bound to tRNA by flexizyme. DBEstands for 3,5-dinitrobenzyl ester and CME stands for cyanomethyl ester.

TABLE 1 Initiator amino acids Acetyl-L-alanine DBEAcetyl-L-phenylalanine CME Acetyl-L-tyrosine CME Acetyl-L-tryptophan CMEAcetyl-D-alanine DBE Acetyl-D-phenylalanine CME Acetyl-D-tyrosine CMEAcetyl-D-tryptophan CME N-Chloroacetyl-L-alanine DBEN-Chloroacetyl-L-phenylalanine CME N-Chloroacetyl-L-tyrosine CMEN-Chloroacetyl-L-tryptophan CME N-Chloroacetyl-D-alanine DBEN-Chloroacetyl-D-phenylalanine CME N-Chloroacetyl-D-tyrosine CMEN-Chloroacetyl-D-tryptophan CME N-3-chloromethylbenzoyl-L-tyrosine CMEN-3-chloromethylbenzoyl-L-tryptophane CME

TABLE 2 Amino acids that crosslink within a peptideNγ-(2-chloroacetyl)-α,γ-diaminobutylic acid DBENγ-(2-chloroacetyl)-α,γ-diaminopropanoic acid DBE

TABLE 3 D-amino acids D-Serine DBE D-Phenylalanine CME D-Tyrosine CMED-Tryptophan CME

TABLE 4 N-methylamino acids N-methyl-Glycine DBE N-methyl-Alanine DBEN-methyl-Serine DBE N-methyl-Histidine DBE N-methyl-Phenylalanine CMEN-methyl-Tyrosine CME N-methyl-Tryptophan CME

TABLE 5 Peptoid blocks N-ethyl-Glycine DBE N-n-propyl-Glycine DBEN-n-butyl-Glycine DBE N-n-pentyl-Glycine DBE N-n-hexyl-Glycine DBEN-n-heptyl-Glycine DBE N-n-octyl-Glycine DBE N-isopentyl-Glycine DBEN-(2-phenylethyl)-Glycine CME N-(3-phenylpropyl)-Glycine CMEN-[2-(p-hydroxyphenyl)ethyl]-Glycine CME

TABLE 6 Other non-canonical amino acids p-biphenylalanine CMEp-trifluoromethylphenylalanine CME p-azidophenylalanine CMEp-biotinyl-aminophenylalanine CME e-N-Biotinyl-lysine DBEe-N-Acetyl-lysine DBE L-Citrulline DBE L-5-Hydroxytryptphan CMEL-1,2,3,4,-Tetrahydroisoquinoline-3-carboxylic acid DBE Aminoisobutyricacid DBE N-methyl-aminoisobutyric acid DBE N-methyl-Phenylglycine CME

The above-described peptide (iii) or (xvii) may be any peptide insofaras it has an amino acid sequence with deletions, additions orsubstitutions of one or several amino acids in any of SEQ ID NOs: 1, 2,and 36 to 44 and can be bound to hemagglutinin. Those skilled in the artcan confirm by a known method whether it can be bound to hemagglutininor not.

The term “peptide with deletions, additions, or substitutions of one orseveral amino acids” as used herein does not limit the number of aminoacids to be deleted, added or substituted insofar as the peptide retainsits binding ability to hemagglutinin. It may be, for example, one tofive, one to three, or one or two. The deletion, addition, orsubstitution position may be either the end or middle of the peptide andthe number of the position may be either one or more.

The peptide (iv) or (xviii) may be any peptide insofar as it has 90% ormore sequence identity to any of SEQ ID NOs: 1, 2, and 36 to 44 and canbind to hemagglutinin. The sequence identity may be 95% or more or 98%or more.

In one aspect, the hemagglutinin-binding peptide of the presentinvention comprises a polypeptide consisting of any of theabove-described amino acid sequences (v) to (vii). Specific examples ofsuch a polypeptide include:

polypeptides having an amino acid sequence with a substitution of anamino acid at position 12 by a hydroxyamino acid in SEQ ID NO: 1;

polypeptides having an amino acid sequence with a substitution of anamino acid at position 13 by a basic amino acid in SEQ ID NO: 1; and

polypeptides having an amino acid sequence with a substitution of anamino acid at position 13 by an amino acid smaller than arginine in SEQID NO: 1.

Examples of the hydroxyamino acid include serine, threonine, tyrosine,hydroxyproline, and hydroxylysine. In one aspect, for example,hydroxyproline can be used for substitution.

Examples of the basic amino acid include arginine, lysine, citrulline,ornithine, creatine, histidine, diaminobutanoic acid, anddiaminopropionic acid. In one aspect, for example, lysine ordiaminopropionic acid can be used for substitution.

The amino acid smaller than arginine is not particularly limited insofaras it is an amino acid having a side chain after substitution smallerthan that of arginine. For example, an amino acid selected from alanine,glutamic acid, and 2,3-diaminopropionic acid can be used forsubstitution.

In another aspect, a hemagglutinin-binding peptide of the presentinvention contains a polypeptide having an amino acid sequencerepresented by the following formula (I):

(SEQ ID NO: 3) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃ (I)(wherein,

Xaa₁ is Ser or Thr,

Xaa₂ is an N-methylamino acid,

Xaa₃ is an arbitrary amino acid,

Xaa₄ is a basic amino acid,

Xaa₅ is Val,

Xaa₆ is a basic amino acid,

Xaa₇ is Tyr,

Xaa₈ is Ser or Thr,

Xaa₉ is Val,

Xaa₁₀ is MePhe,

Xaa₁₁ is Asn,

Xaa₁₂ is MeAla, and

Xaa₁₃ is Val or Ser.

Xaa₂ may be MePhe or MeGly,

Xaa₃ may be MeGly or Thr,

Xaa₄ may be His, and

Xaa₆ may be His or Arg.

Further, the polypeptide represented by the formula (I) may beSer-MePhe-MeGly-His-Val-His-Tyr-Ser-Val-MePhe-Asn-MeAla-Val (SEQ ID NO:4) or

Thr-MeGly-Thr-His-Val-Arg-Tyr-Thr-Val-MePhe-Asn-MeAla-Ser (SEQ ID NO:5).

In the formula, MeAla represents N-methylalanine.

The hemagglutinin-binding peptide of the present invention may be apeptide consisting of only an amino acid sequence of any of (i) to(xviii) or a peptide consisting of only an amino acid sequence of theformula (I); or a polypeptide having one or more amino acids bound to atleast one end thereof. The full length of the hemagglutinin-bindingpeptide of the present invention can be adjusted to, for example, 30amino acids or less, 20 amino acids or less, or 15 amino acids or less.

The peptide of the present invention embraces various derivativesthereof insofar as they can achieve the object of the present invention.Examples of the derivatives include derivatives having an amide, ester,or carboxyl group as the C-terminus thereof and the peptides fused witha cell-penetrating peptide (CPP) to facilitate introduction of thepeptide into the cell when they are administered. CPP is a generic nameof peptides having affinity for cell membranes and transition propertyinto cells. Some of the known CCPs are protein-transduction domain (PTD)which is a domain consisting of 11 amino acids of Trans-activator oftranscription protein (TAT protein) in which HIV-virus is expressed,Antennapedia of Drosophila, VP22 derived from Herpes virus,oligoarginine, and penetratin. In general, CPP tends to have a highcontent of a basic amino acid such as arginine, lysine, or histidine.

Additional examples of the derivatives of the peptide of the presentinvention include those obtained by modification such asphosphorylation, methylation, acetylation, adenylylation,ADP-ribosylation, or glycosylation and fused protein obtained by fusionwith another peptide or protein. These derivatives can be prepared bythose skilled in the art in a known manner or a method based thereon.

The hemagglutinin-binding peptide of the present invention embracessalts thereof insofar as they can achieve the object of the presentinvention. As the salts of the peptide, salts with physiologicallyacceptable base or acid are used. Examples include addition salts withan inorganic acid (such as hydrochloric acid, hydrobromic acid,hydroiodic acid, sulfuric acid, or phosphoric acid), addition salts withan organic acid (such as p-toluenesulfonic acid, methanesulfonic acid,oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid,citric acid, benzoic acid, or acetic acid), inorganic bases (such asammonium hydroxide, alkali or alkaline earth metal hydroxide, carbonate,or bicarbonate), and an amino acid.

The hemagglutinin-binding peptide of the present invention may becyclized (macrocyclized). The term “cyclized” as used herein means thattwo amino acids apart from each other by at least one amino acid binddirectly or bind indirectly via a linker or the like to each other inone peptide to form a cyclic structure in the molecule.

Cyclization may be achieved via a disulfide bond, peptide bond, alkylbond, alkenyl bond, ester bond, thioester bond, ether bond, thioetherbond, phosphate ether bond, azo bond, C—S—C bond, C—N—C bond, C═N—Cbond, amide bond, lactam bridge, carbamoyl bond, urea bond, thioureabond, amine bond, thioamide bond, or the like, but not limited to them.

A cyclization of a peptide sometimes stabilizes the peptide structureand thereby enhance affinity for a target.

As amino acids for macrocyclization, for example, an amino acid havingthe following functional group 1 and an amino acid having a functionalgroup 2 corresponding thereto can be used. Either the functional group 1or the functional group 2 may be placed on the N-terminal side. Theamino acid having the functional group 1 and the amino acid having thefunctional group 2 may each be an N-terminal amino acid or C-terminalamino acid or a non-terminal amino acid.

TABLE 7 Functional group 1 Functional group 2 (A)

HS— (A-2) (B) —C≡C—H N₃— (B-1) (B-2) (C) —Ar—CH₂NH₂ (C-1)

(D) —C≡C—CH₂—X₁ HS— (D-1) (D-2) (E) —Ar—CH₂—X₁ HS— (E-1) (E-2)

In the above formulas, X₁ represents CI, Br, or I and Ar represents asubstituted or unsubstituted aromatic ring.

As the amino acid (A-1), for example, a chloroacetylated amino acid canbe used. Examples of the chloroacetylated amino acid includeN-chloroacetyl-L-alanine, N-chloroacetyl-L-phenylalanine,N-chloroacetyl-L-tyrosine, N-chloroacetyl-L-tryptophan,N-3-(2-chloroacetamido)benzoyl-L-phenylalanine,N-3-(2-chloroacetamido)benzoyl-L-tyrosine,N-3-(2-chloroacetamido)benzoyl-L-tryptophane,β-N-chloroacetyl-L-diaminopropanoic acid,γ-N-chloroacetyl-L-diaminobutyric acid, σ-N-chloroacetyl-L-ornithine,ε-N-chloroacetyl-L-lysine, N-3-chloromethylbenzoyl-L-tyrosine, andN-3-chloromethylbenzoyl-L-tryptophane and D-amino acid derivativescorresponding thereto (for example, N-Chloroacetyl-D-alanine,N-Chloroacetyl-D-phenylalanine, N-Chloroacetyl-D-tyrosine, andN-Chloroacetyl-D-tryptophan).

Examples of the amino acid (A-2) include cysteine, homocysteine,mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid,2-amino-8-mercaptooctanoic acid, and amino acids obtained by protectingthe SH group of these amino acids and then eliminating the protectinggroup, and D-amino acid derivatives corresponding thereto.

The cyclization method can be carried out, for example, according to themethod described in Kawakami, T. et al., Nature Chemical Biology 5,888-890 (2009); Yamagishi, Y. et al., ChemBioChem 10, 1469-1472 (2009);Sako, Y. et al., Journal of American Chemical Society 130, 7932-7934(2008); Goto, Y. et al., ACS Chemical Biology 3, 120-129 (2008); orKawakami T. et al, Chemistry & Biology 15, 32-42 (2008), orWO2008/117833.

As the amino acid (B-1), for example, propargylglycine,homopropargylglycine, 2-amino-6-heptynoic acid, 2-amino-7-octynoic acid,and 2-amino-8-nonynoic acid can be used. In addition, 4-pentynoylated or5-hexynoylated amino acids can also be used. Examples of the4-pentynoylated amino acids include N-(4-pentenoyl)-L-alanine,N-(4-pentenoyl)-L-phenylalanine, N-(4-pentenoyl)-L-tyrosine,N-(4-pentenoyl)-L-tryptophan,N-3-(4-pentynoylamido)benzoyl-L-phenylalanine,N-3-(4-pentynoylamido)benzoyl-L-tyrosine,N-3-(4-pentynoylamido)benzoyl-L-tryptophane,β-N-(4-pentenoyl)-L-diaminopropanoic acid,γ-N-(4-pentenoyl)-L-diaminobutyric acid, σ-N-(4-pentenoyl)-L-ornithine,and ε-N-(4-pentenoyl)-L-lysine, and D-amino acid derivativescorresponding thereto.

As the amino acid (B-2), for example, azidoalanine,2-amino-4-azidobutanoic acid, azidoptonorvaline, azidonorleucine,2-amino-7-azidoheptanoic acid, and 2-amino-8-azidooctanoic acid can beused. In addition, azidoacetylated or 3-azidopentanoylated amino acidscan also be used. Examples of the azidoacetylated amino acids includeN-azidoacetyl-L-alanine, N-azidoacetyl-L-phenylalanine,N-azidoacetyl-L-tyrosine, N-azidoacetyl-L-tryptophan,N-3-(4-pentynoylamido)benzoyl-L-phenylalanine,N-3-(4-pentynoylamido)benzoyl-L-tyrosine,N-3-(4-pentynoylamido)benzoyl-L-tryptophane,β-N-azidoacetyl-L-diaminopropanoic acid,γ-N-azidoacetyl-L-diaminobutyric acid, σ-N-azidoacetyl-L-ornithine, andε-N-azidoacetyl-L-lysine, and D-amino acid derivatives correspondingthereto.

The cyclization method can be performed, for example, according to themethod described in Sako, Y. et al., Journal of American ChemicalSociety 130, 7932-7934 (2008) or WO2008/117833.

Examples of amino acid (C-1) includeN-(4-aminomethyl-benzoyl)-phenylalanine (_(AMB)F) and4-3-aminomethyltyrosine.

Examples of the amino acid (C-2) include 5-hydroxytryptophan (W_(OH)).

The cyclization method can be performed, for example, according to themethod described in Yamagishi, Y. et al., ChemBioChem 10, 1469-1472(2009) or WO2008/117833.

Examples of the amino acid (D-1) include 2-amino-6-chloro-hexynoic acid,2-amino-7-chloro-heptynoic acid, and 2-amino-8-chloro-octynoic acid.

Examples of the amino acid (D-2) include cysteine, homocysteine,mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid,and 2-amino-8-mercaptooctanoic acid, amino acids obtained by protectingthe SH group of these amino acids and then eliminating the protectinggroup, and D-amino acid derivatives corresponding thereto.

The cyclization method can be performed, for example, according to themethod described in WO2012/074129.

Examples of the amino acid (E-1) includeN-3-chloromethylbenzoyl-L-phenylalanine,N-3-chloromethylbenzoyl-L-tyrosine, andN-3-chloromethylbenzoyl-L-tryptophane.

Examples of the amino acid (E-2) include cysteine, homocysteine,mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid,and 2-amino-8-mercaptooctanoic acid, and amino acids obtained byprotecting the SH group of these amino acids and then eliminating theprotecting group, and D-amino acid derivatives corresponding thereto.

The amino acids (A-1) to (E-2) can be introduced into thehemagglutinin-binding peptide in a known manner by chemical synthesis ortranslation and synthesis described later.

In one aspect of the present invention, use of the amino acid (A-1) or(A-2), among the above-described amino acids (A-1) to (E-2), forfunctionalization is preferred.

For example, the hemagglutinin-binding peptide of the present inventioncan contain a cyclic peptide consisting of the following amino acidsequence:

(I)  (SEQ ID NO: 10) Cyclo(Ac-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Arg-Cys)-amide;

(II) an amino acid sequence with deletions, additions, or substitutionsof one or several amino acids at a position selected from positions 4and 7 and positions 13 and 14 in SEQ ID NO: 10;

(III) an amino acid sequence with substitutions of one or several aminoacids at a position selected from positions 4, 7, and 14 in SEQ ID NO:10;

(IV) an amino acid sequence with substitution of an amino acid atposition 14 in SEQ ID NO: 10; and

(V) an amino acid sequence of any of SEQ ID NOs: 26 to 35.

In the present specification, hemagglutinin is an antigenic glycoproteinfound on the surface of many bacteria or viruses including influenzavirus and is represented by “HA”. Hemagglutinin is involved in bindingprocedure of a virus to a host cell. More specifically, whenhemagglutinin on the virus surface binds to a target sialic acid on thesurface of a host cell, the virus is wrapped in a cell membrane and isincorporated into the cell in the form of a virus-containing endosome.Then, fusion between an endosome membrane and a viral membrane occursand a viral genome is inserted in the cell, which starts proliferation.

Hemagglutinin has at least 16 sub-types and they are called “H1 to H16”,respectively. The letter H in the subtype name of influenza stands forhemagglutinin.

The peptide of the present invention can be prepared by a known peptidepreparation method, for example, chemical synthesis method such asliquid-phase method, solid-phase method, or hybrid method using aliquid-phase method and a solid-phase method in combination; or generecombination method.

In solid-phase method, an esterification reaction is performed, forexample, between the hydroxyl group of a hydroxyl-containing resin andthe carboxyl group of a first amino acid (usually, C-terminal amino acidof an intended peptide) having an α-amino group protected with aprotecting group. As the esterifying catalyst, a known dehydrationcondensation agent such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole(MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide(DIPCDI) may be used.

Next, the protecting group of the α-amino group of the first amino acidis eliminated and at the same time, a second amino acid having all thefunctional groups protected except the main chain carboxyl group isadded to activate the carboxyl group and bind the first and second aminoacids to each other. Then, the α-amino group of the second amino acid isdeprotected, a third amino acid having all the functional groupsprotected except the main chain carboxyl group is added, and thecarboxyl group is activated to bind the second and third amino acids toeach other. The above-described reactions are repeated to synthesize apeptide having an intended length. Then, all the functional groups aredeprotected.

Examples of the resin for solid-phase synthesis include Merrifieldresin, MBHA resin, CI-Trt resin, SASRIN resin, Wang resin, Rink amideresin, HMFS resin, Amino-PEGA resin (Merck), and HMPA-PEGA resin(Merck). These resins may be provided for use after washed with asolvent (dimethylformamide (DMF), 2-propanol, methylene chloride, or thelike).

Examples of the protecting group of the α-amino group include abenzyloxycarbonyl (Cbz or Z) group, a tert-butoxycarbonyl (Boc) group, afluorenylmethoxycarbonyl (Fmoc) group, a benzyl group, an allyl group,and an allyloxycarbonyl (Alloc) group.

The Cbz group can be deprotected using hydrofluoric acid, hydrogenation,or the like; the Boc group can be deprotected using trifluoroacetic acid(TFA); and the Fmoc group can be deprotected by the treatment withpiperidine.

For protection of the α-carboxyl group, a methyl ester, an ethyl ester,a benzyl ester, a tert-butyl ester, a cyclohexyl ester, or the like maybe used.

As other functional groups of an amino acid, the hydroxyl group ofserine or threonine can be protected with a benzyl group or a tert-butylgroup and the hydroxyl group of tyrosine can be protected with a2-bromobenzyloxycarbonyl group or a tert-butyl group. The amino group ofa lysine side chain or the carboxyl group of glutamic acid or asparticacid can be protected in a manner similar to the α-amino group orα-carboxyl group.

The carboxyl group can be activated with a condensation agent. Examplesof the condensation agent include dicyclohexylcarbodiimide (DCC),diisopropylcarbodiimide (DIPCDI),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC),(1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate (BOP), and1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxidehexafluorophosphate (HBTU).

A peptide chain can be cleaved from the resin by treating it with anacid such as TFA or hydrogen fluoride (HF).

Peptide preparation based on the gene recombination method (translationand synthesis system) can be performed using a nucleic acid encoding thepeptide of the present invention. The nucleic acid encoding the peptideof the present invention may be either DNA or RNA.

The nucleic acid encoding the peptide of the present invention can beprepared in a known manner or based thereon. For example, it can besynthesized using an automated synthesizer. The DNA thus obtained mayhave therein a restriction enzyme recognition site for inserting it intoa vector or may have therein a base sequence that encodes an amino acidsequence for cleavage of the resulting peptide chain by an enzyme.

As described above, when the peptide of the present invention is fusedwith a membrane permeant peptide, the nucleic acid contains a nucleicacid encoding the membrane permeant peptide.

In order to suppress decomposition by a host-derived protease, a chimeraprotein expression method that expresses the intended peptide as achimera peptide with another peptide can be used. In this case, as thenucleic acid, a nucleic acid encoding the intended peptide and a peptidethat binds thereto is used.

Then, an expression vector is prepared using the nucleic acid encodingthe peptide of the present invention. The nucleic acid can be insertedinto downstream of a promoter of an expression vector as it is, or afterdigestion with a restriction enzyme or addition of a linker. Examples ofthe vector include Escherichia coli-derived plasm ids (such as pBR322,pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, and pBluescript II),Bacillus subtilis-derived plasmids (such as pUB110, pTP5, pC1912, pTP4,pE194, and pC194), yeast-derived plasmids (such as pSH19, pSH15, YEp,YRp, Ylp, and YAC), bacteriophages (such as e phage and M13 phage),viruses (retrovirus, vaccinia virus, adenovirus, adeno-associated virus(AAV), cauliflower mosaic virus, tobacco mosaic virus, and baculovirus),and cosmids.

The promoter can be selected as needed, depending on the type of thehost. When the host is an animal cell, for example, a SV40 (simian virus40)-derived promoter or a CMV (cytomegalovirus)-derived promoter can beused. When the host is Escherichia coli, a trp promoter, a T7 promoter,a lac promoter, or the like can be used.

The expression vector may incorporate therein a nucleic acid encoding aDNA replication origin (ori), a selection marker (antibiotic resistance,nutrition requirement, or the like), an enhancer, a splicing signal, apolyadenylation signal, a tag (FLAG, HA, GST, GFP, or the like), or thelike.

Next, an appropriate host cell is then transformed using theabove-described vector. The host can be selected as needed based on therelation with a vector and for example, Escherichia coli, Bacillussubtilis, Bacillus bacteria), yeasts, insects or inset cells, and animalcells can be used. Examples of the animal cells include HEK293T cells,CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells.Transformation can be performed in a known manner such as lipofection,calcium phosphate method, electroporation, microinjection, or particlegun technology, depending on the type of hosts. By culturing thetransformant in a conventional manner, an intended peptide is expressed.

The peptide from the cultured product of the transformant can bepurified in the following manner. Cultured cells collected and thensuspended in an appropriate buffer are destructed by ultrasonictreatment, freezing and thawing method, or the like and the resultingdestructed product centrifuged or filtered to obtain a crude extract.When the peptide is secreted in the culture fluid, a supernatant iscollected.

Purification of the crude extract or culture supernatant can also beperformed by a known method or a method based thereon (for example,salting-out, dialysis, ultrafiltration, gel filtration, SDS-PAGE, ionexchange chromatography, affinity chromatography, or reverse-phasehigh-performance liquid chromatography).

The peptide thus obtained may be converted from a free peptide to a saltthereof or from a salt thereof to a free peptide by a known method or amethod based thereon.

The system for translation and synthesis may be a cell-free translationsystem. The cell-free translation system may include, for example, alibosome protein, aminoacyl tRNA synthetase (ARS), ribosome RNA, anamino acid, rRNA, GTP, ATP, a translation initiation factor (IF), anelongation factor (EF), a release factor (RF), a ribosome regenerationfactor (RRF), and other factors necessary for translation. AnEscherichia coli extract or wheat bran extract may be added in order toincrease the expression efficiency. Further, a rabbit erythrocyteextract or insect cell extract may be added.

Continuous energy supply to a system containing the above by dialysisenables production of several hundred μg to several mg/mL of a protein.The system may contain RNA polymerase for carrying out transcriptionfrom DNA at the same time. As a commercially available cell-freetranslation system, an Escherichia-coli derived system such as“RTS-100™” of Roche Diagnostics Corporation or PURESYSTEM™ of PGICorporation or a system using wheat germ extract such as that of ZOEGENECorporation or Cell-free Science may be used.

By using the cell-free translation system, a high-purity peptide can beobtained without purifying the expression product.

In the cell-free translation system, an artificial aminoacyl tRNAobtained by linking (acylating) a desired amino acid or hydroxy acid totRNA may be used instead of an aminoacyl tRNA synthesized by a nativeaminoacyl tRNA synthetase. Such an aminoacyl tRNA can be synthesizedusing an artificial ribozyme.

Examples of such a ribozyme include flexizymes (H. Murakami, H. Saito,and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; H. Murakami,D. Kourouklis, and H. Suga, (2003), Chemistry & Biology, Vol. 10,1077-1084; H. Murakami, A. Ohta, H. Ashigai, H. Suga (2006) NatureMethods 3, 357-359 “The flexizyme system: a highly flexible tRNAaminoacylation tool for the synthesis of nonnatural peptides”; N. Niwa,Y. Yamagishi, H. Murakami, H. Suga (2009) Bioorganic & MedicinalChemistry Letters 19, 3892-3894 “A flexizyme that selectively chargesamino acids activated by a water-friendly Leaving group”; WO2007/066627;and the like). Flexizyme is also known as, as well as flexizyme (Fx) inoriginal form, dinitrobenzyl flexizyme (dFx), enhanced flexizyme (eFx),or aminoflexizyme (aFx), each obtained by modifying the original one.

By using a tRNA having a desired amino acid or hydroxy acid linkedthereto and prepared using flexizyme, a desired codon can be translatedwhile associating the codon with the desired amino acid or hydroxy acid.As the desired amino acid, a non-canonical amino acid may be used. Forexample, a non-natural amino acid necessary for the above-describedcyclization can be introduced into the hemagglutinin-binding peptide bythis method.

(Pharmaceutical Composition and Treatment Method)

As shown later in Examples, the peptide of the present invention bindsto hemagglutinin on the surface of an influenza virus and therebyexhibits an anti-influenza virus effect. A composition containing thepeptide of the present invention is therefore useful as a preventive ortreatment agent of influenza. Similarly, the peptide of the presentinvention is useful in a prevention or treatment method of influenza.

The term “influenza” as used herein means an acute infection caused byan influenza virus. When humans are infected, they have cold-likesymptoms with high fever or muscle pain. They sometimes have agastrointestinal symptom such as stomachache, vomiting, or diarrhea.Complications include pneumonia and influenza encephalopathy.

The term “infection” as used herein means either one of a process of avirus entering the living body via a skin or mucous membrane or aprocess of a virus entering a cell by membrane fusion. The term “viralinfection” as used herein means a state that a virus has entered theliving body regardless of the presence or absence of symptoms.

In the present specification, the term “treatment or prevention ofinfluenza” is used in its widest meaning. For example, it meansalleviation of one or more symptoms associated with infection with aninfluenza virus or prevention of worsening of them, suppression ofoccurrence of symptoms after infection, inhibition (retardation ortermination) of viral infection of cells in vivo, inhibition(retardation or termination) of proliferation of a virus in vivo, ordecreasing of the number of viruses in vivo. The treatment or preventionof influenza exhibiting at least one of the above-described effects isdetermined useful.

As shown later in Examples, the peptide of the present invention hashemagglutinin neutralizing activity and thus, it is recognized as havingan effect similar to that of an influenza vaccine.

In the present specification, the administration route of thepharmaceutical composition is not particularly limited and it may beadministered either orally or parenterally. Examples of the parenteraladministration include administration by injection such asintramuscular, intravenous, or subcutaneous injection, transdermaladministration, and transmucosal administration (nasal, buccal, ocular,pulmonary, vaginal, or rectal).

Since the polypeptide in the pharmaceutical composition is readilymetabolized and excreted, it can be subjected to various modifications.For example, a polypeptide can have longer retention time in blood andreduced antigenicity by adding thereto polyethylene glycol (PEG) orsugar chain. A polypeptide may be encapsulated using a sustained-releasebase such as an emulsion, nanoparticles, nanospheres, or the likeprepared from a biodegradable polymer compound (such as polylactic acidglycol (PLGA)), porous hydroxyapatite, liposome, surface-modifiedliposome, or unsaturated fatty acid. When it is administeredtransdermally, it can be penetrated through the stratum corneum bypassing a weak electrical current through the skin surface(iontophoresis).

As the pharmaceutical composition, the active ingredient may be used asis or a formulated by adding thereto a pharmaceutically acceptablecarrier, excipient, additive, or the like. Examples of the dosage forminclude solutions (for example, injections), dispersions, suspensions,tablets, pills, powdered drug, suppositories, powders, fine granules,granules, capsules, syrups, troches, inhalants, ointments, ophthalmicformulations, nasal formulations, ear formulations, and cataplasms.

The formulation can be obtained in a conventional manner by using, forexample, an excipient, a binder, a disintegrant, a lubricant, adissolving agent, a solubilizing agent, a colorant, a taste/odorcorrigent, a stabilizer, an emulsifier, an absorption promoter, asurfactant, a pH regulator, an antiseptic, or an antioxidant as needed.

Examples of the ingredient to be used for obtaining the formulationinclude, but not limited to, purified water, saline, phosphate buffer,pharmaceutically acceptable organic solvents such as dextrose, glycerol,and ethanol, animal or vegetable oils, lactose, mannitol, glucose,sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, cornstarch, silicic anhydride, magnesium aluminum silicate, collagen,polyvinyl alcohol, polyvinyl pyrrolidine, carboxy vinyl polymer,carboxymethylcellulose sodium, sodium polyacrylate, sodium alginate,water-soluble dextran, carboxymethyl starch sodium, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum arabic, tragacanth, casein,agar, polyethylene glycol, diglycerin, glycerin, polypropylene glycol,petrolatum, paraffin, octyl dodecyl myristate, isopropyl myristate,higher alcohol, stearyl alcohol, stearic acid, and human serum albumin.

Since peptides have difficulty in transmucosal absorption, theabove-described pharmaceutical composition may contain an absorptionpromoter for improving absorption of a poorly absorbable drug. Examplesof such an absorption promoter include surfactants such aspolyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponin; bilesalts such as glycocholate, deoxycholate, and taurocholate; chelatingagents such as EDTA and salicylic acid; fatty acids such as caproicacid, capric acid, lauric acid, oleic acid, linoleic acid, and mixedmicelle; enamine derivatives, N-acylcollagen peptide, N-acylaminoicacid, cyclodextrines, chitosans, and nitric oxide donors.

Pills or tablets may be sugar-, gastric-, or enteric-coated.

Injections may contain distilled water for injection, physiologicalsaline, propylene glycol, polyethylene glycol, a vegetable oil, analcohol, or the like. It may further contain a humectant, an emulsifier,a dispersant, a stabilizer, a dissolving agent, a solubilizing agent, anantiseptic, or the like.

The dose of the pharmaceutical composition of the present invention whenadministered to mammals (for example, humans, mice, rats, guinea pigs,rabbits, dogs, horses, monkeys, and pigs), particularly, humans differsdepending on the symptom, age, sex, weight, difference in sensitivity ofpatients, administration method, administration interval, type of theactive ingredient, and type of the formulation and is not particularlylimited. For example, from 30 μg to 100 g, from 100 μg to 500 mg, orfrom 100 μg to 100 mg can be administered once or in several portions.When it is administered by injection, from 1 μg/kg to 3000 μg/kg or from3 μg/kg to 1000 μg/kg may be administered once or in several portions,depending on the weight of a patient.

The prevention or treatment method of influenza using the peptide of thepresent invention can be performed referring to the above descriptionrelating to the pharmaceutical composition.

(Influenza Virus Detection Agent and Detection Kit)

The present invention also embraces an influenza virus detection agentcontaining the peptide of the present invention. The peptide of thepresent invention specifically binds to hemagglutinin on the surface ofan influenza virus. The influenza virus in a sample can therefore bydetected using the peptide of the present invention instead of, forexample, an anti-influenza antibody in immunoassay such as ELISA.

When the peptide of the present invention is used as a detection agent,it may be detectably labeled. For labeling of the peptide, for example,an antibody labeled with: an enzyme such as peroxidase or alkalinephosphatase; a radioisotope such as ¹²⁵I, ¹³¹I, ³⁵S, or ³H; afluorescent substance such as fluorescein isothiocyanate, rhodamine,dansyl chloride, phycoerythrin, tetramethyl rhodamine isothiocyanate, ornear infrared fluorescent material; a light-emitting substance such asluciferase, luciferin, or aequorin. In addition, an antibody labeledwith nanoparticles such as gold colloid or quantum dot can also bedetected.

In immunoassay, detection can also be achieved by labeling the peptideof the present invention with biotin and then binding avidin orstreptavidin labeled with an enzyme or the like to the peptide.

Among immunoassays, ELISA using enzyme labeling is preferred because anantigen can be measured conveniently and rapidly. For example, aninfluenza virus can be detected by immobilizing an antibody thatspecifically recognizes a moiety of an influenza virus other thanhemagglutinin onto a solid-phase support, adding a sample to react thesame, adding the peptide of the present invention which has been labeledto react the same, and, washing, performing reaction with an enzymesubstrate, followed by color development, and absorbance measurement. Itis also possible to, after the reaction between the sample and theantibody immobilized onto a solid phase support, add the peptide of thepresent invention which has not been labeled, and add an antibodyagainst the peptide of the present invention labeled with an enzyme.

When the enzyme is a peroxidase, 3,3′-diaminobenzidine (DAB),3,3′5,5′-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), or thelike can be used as the enzyme substrate. When the enzyme is an alkalinephosphatase, p-nitrophenyl phosphate (NPP) or the like can be used.

The “solid phase support” described herein is not particularly limitedinsofar as it permits immobilization of an antibody thereonto. Examplesinclude microtiter plates, substrates, and beads made of glass, a metal,a resin, or the like, nitrocellulose membranes, nylon membranes, andPVDF membranes. The target substance can be immobilized onto such asolid phase support in a known manner.

The detection kit of the present invention may further include a reagentand a tool necessary for the detection (the peptide of the presentinvention, an antibody, a solid-phase support, a buffer, an enzymaticreaction quenching solution, a microplate reader, and the like but notlimited to them).

EXAMPLES

Examples described below are merely for exemplary purpose and theymerely intend to describe the above embodiments and the presentinvention in detail. They do not limit the present invention.

Example 1

[Chemical Synthesis of Cyclic Peptide that Binds to Hemagglutinin [1]]

A hemagglutinin-binding peptide was identified by a screening methodsimilar to that described in Patent Document 3. The peptide waschemically synthesized in order to confirm whether it has bindingactivity to hemagglutinin and whether it has inhibition activity againstproliferation of an influenza virus.

Peptide synthesis was performed in accordance with a common solid-phasesynthesis method using: 9-fluorenylmethoxycarbonyl group (Fmoc) as aprotecting group of an α amino group; and an automated synthesizer SyroI (product of Biotage Japan).

Rink Amide AM resin (product of Novabiochem) was added in an amountcorresponding to 3 μmol to a synthesis chip and2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) and Fmoc-protected amino acid (each, product of Novabiochem) werereacted, successively.

After N-terminal tryptophan was linked and Fmoc was removed, 6equivalents of chloroacetyl N-hydroxysuccinate dissolved in1-methyl-2-pyrrolidinone (NMP; product of Nacalai Tesuque) were addedand the resulting mixture was allowed to stand for one hour.

The peptide resin thus obtained was washed three times withN,N-dimethylformamide (DMF: product of Nacalai Tesuque) and methylenechloride and then dried. A trifluoroacetic acid-water-triisopropylsilanemixture (90:5:5 by volume) was added and the resulting mixture wasstirred at room temperature for 2 hours.

The crude peptide cleaved was collected by ether precipitation. Afterwashing twice with diethyl ether and drying, an 80% aqueous DMSOsolution containing 125 mM tris-hydroxymethylaminomethane was added togive a final concentration of 10 mM, followed by stirring at roomtemperature for one hour.

The cyclic peptide solution was acidified, filtered, and then, purifiedby reverse-phase high-performance liquid chromatography using a C4column (Cosmosil AR300 5C4 (10×250 mm, product of Nacalai Tesuque) toobtain an intended product. Water-acetonitrile containing 0.1%trifluoroacetic acid was used as a mobile phase. The peptide thusobtained was identified by MALDI-TOF-MS using Autolex II (product ofBruker Daltonics).

The following four peptides were synthesized:

iHA-100 (SEQ ID NO: 6)^(ClAc)-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Arg-Cys-amide; iHA-101 (SEQ ID NO: 7)^(ClAc)-Trp-Arg-Val-Ser-MePhe-Thr-Tyr-MePhe-MeSer-Tyr-Thr-Pro-Ser-Cys-amide; iHA-102 (SEQ ID NO: 8)^(ClAc)-Trp-Ser-MePhe-MeGly-His-Val-His-Tyr-Ser-Val-MePhe-Asn-MeAla-Val-Cys-amide;  or iHA-103 (SEQ ID NO: 9)^(ClAc)-Trp-Thr-MeGly-Thr-His-Val-Arg-Tyr-Thr-Val-MePhe-Asn-MeAla-Ser-Cys-amide.

The following peptides obtained by cyclization of the above fourpeptides were used in the growth inhibition test of an influenza virus.

iHA-100 (SEQ ID NO: 10)Cyclo(Ac-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Arg-Cys)-amide; iHA-101 (SEQ ID NO: 11)Cyclo(Ac-Trp-Arg-Val-Ser-MePhe-Thr-Tyr-MePhe-MeSer-Tyr-Thr-Pro-Ser-Cys)-amide; iHA-102 (SEQ ID NO: 12)Cyclo(Ac-Trp-Ser-MePhe-MeGly-His-Val-His-Tyr-Ser-Val-MePhe-Asn-MeAla-Val-Cys)-amide;  or iHA-103 (SEQ ID NO: 13)Cyclo(Ac-Trp-Thr-MeGly-Thr-His-Val-Arg-Tyr-Thr-Val-MePhe-Asn-MeAla-Ser-Cys)-amide.

As a positive control, the following iHA-24 cyclic peptide (PatentDocument 3) was used.

iHA-24 (linear) (SEQ ID NO: 14)^(ClAc)-Trp-Trp-Leu-Asp-Pro-Tyr-Trp-Leu-Thr-Trp-Tyr- Thr-Cys-Gly-amideiHA-24 (cyclic) (SEQ ID NO: 15)Cyclo(Ac-Trp-Trp-Leu-Asp-Pro-Tyr-Trp-Leu-Thr-Trp- Tyr-Thr-Cys)-Gly-amide

Example 2

[Evaluation of Peptide Activity Against Influenza Virus [1]]

1. Neutralization Activity

In order to confirm the neutralization activity of peptides against aninfluenza virus, a test was performed by mixing each peptide and theinfluenza virus first and then bringing the resulting mixed solutioninto contact with cells. The following is a specific test method.

1) Three days before assay, a 6-well plate was seeded with 2×10⁵cells/well of MDCK cells.

2) One day before infection with the influenza virus, the cells werepre-treated by exchanging cell culture media with those containing 0.01μM, 0.1 μM, and 1 μM of each of the peptides.

3) The respective cell culture media containing 0.01 μM, 0.1 μM, and 1μM of each of the peptides were mixed with 110 μl of a 55PFU virussolution and the resulting mixture was incubated at 37° C. for one hourin a CO₂ incubator.

4) To each well was added 0.2 ml of each of the virus solutions obtainedby incubation with the peptide and the solution was delivered throughoutthe plate.

5) The virus was allowed to adsorb to the peptide at 37° C. or 34° C.for one hour and the plate was shaken every 15 minutes.

6) The virus solution was removed, followed by washing once withBSA(−)/MEM.

7) Each well was overlaid with 2 ml of 1% BSA/MEM added with 0.8%agarose.

8) After solidification of agarose, the plate was turned upside down andplaced in a CO₂ incubator.

9) The cells were cultured for 2 or 3 days at 37° C. or 34° C.

10) 10% Formalin was added and the cells were fixed at room temperaturefor one hour.

11) The formalin and agarose medium were removed, followed by washingwith water.

12) The cells were stained with 1% crystal violet.

13) After washing with water and drying, the number of plaques wascounted.

2. Neutralization+Inhibition Activity

In addition to the evaluation of the neutralization activity by theabove-described method, growth inhibition activity was evaluated byadding the peptide or an anti-influenza drug to an agarose gel. Thefollowing is a specific test method.

1) Three days before assay, a 6-well plate was seeded with 2×10⁵cells/well of MDCK cells.

2) One day before infection with the influenza virus, the cells werepre-treated by exchanging cell culture media with those containing 0.01μM, 0.1 μM, and 1 μM of each of the peptides.

3) The respective cell culture media containing 0.01 μM, 0.1 μM, and 1μM of each of the peptides were mixed with 110 μl of a 55PFU virussolution and the resulting mixture was incubated at 37° C. for one hourin a CO₂ incubator.

4) To each well was added 0.2 ml of each of the virus solutions obtainedby incubation with the peptide and the solution was delivered throughoutthe plate.

5) The virus was allowed to adsorb to the peptide at 37° C. or 34° C.for one hour and the plate was shaken every 15 minutes.

6) The virus solution was removed, followed by washing once withBSA(−)/MEM.

7) Each well was overlaid with 2 ml of 1% BSA/MEM added with 0.01 μM,0.1 μM, or 1 μM of the peptide, 10 μg/ml acetyltrypsin, and 0.8%agarose. Alternatively, each well was overlaid with an agarose mediumcontaining 0.01 μM, 0.1 μM, or 1 μM of oseltamivir (Tamiflu, trade name)or zanamivir (Relenza, trade name).8) After solidification of agarose, the plate was turned upside down andplaced in a CO₂ incubator.9) The cells were cultured for 2 or 3 days at 37° C. or 34° C.10) 10% Formalin was added and the cells were fixed at room temperaturefor one hour.11) The formalin and agarose medium were removed, followed by washingwith water.12) The cells were stained with 1% crystal violet.13) After washing with water and drying, the number of plaques wascounted.3. Results3-1. Results of Evaluation of the Activity of Peptides Against InfluenzaVirus H5N1 Vac-3

FIG. 1 shows the evaluation results of the neutralization activity andneutralization+growth inhibition activity of peptides while using H5N1Vac-3 as an influenza virus. In any of the peptides iHA-100 to 103, thenumber of plaques showed a dose-dependent decrease. The anti-viraleffect of the peptides iHA-100, 102, and 103 was markedly higher thanthat of the peptide iHA-24, a positive control, and the effect wascomparable to that of zanamivir (Relenza; trade name).

Confirmation by back-titration (Back in this drawing) has revealed thatthe amount of the virus used was a predetermined virus amount.

3-2. Results of Evaluation of the Activity of Peptides Against HighlyPathogenic H5N1 Strain

The following three viruses were used as the highly pathogenic avianinfluenza virus. FIG. 2 shows the evaluation results of theneutralization activity and neutralization activity+growth inhibitionactivity of peptides.

A/ws/Hokkaido/1/08 (clade 2.3)

A/ws/Mongolia/3/05 (clade 2.2)

A/Vietnam/UT3040/04 (clade 1)

The effect of iHA-100 against the highly pathogenic avian influenzavirus strain was also markedly higher than that of iHA-24 and is on thesame level as that of zanamivir.

Confirmation by back-titration (“Back” in this figure) has revealed thatthe amount of the virus used was a predetermined virus amount.

3-3. Results of Evaluation of the Activity of Peptides Against H1N1-Pdm2619 Strain, One of Isolated Strains of Influenza Virus H1N1-Pdm 2009

FIG. 3 shows the evaluation results of neutralization activity andneutralization+growth inhibition activity of peptide against H1N1-pdm2619 used as an influenza virus.

When iHA-100 to 103 were used, the number of plaques dose-dependentlydecreased in a manner equal to or higher than that of iHA-24 and inparticular, iHA-100 exhibited an anti-viral effect comparable to that ofzanamivir (Relenza, trade name)

3-4. Inhibition and Neutralization Activity Against Influenza VirusH2N2-Adachi Strain

Similarly, FIG. 4 shows the evaluation results of neutralizationactivity and neutralization+growth inhibition activity of peptidesagainst influenza virus H2N2-Adachi strain.

Compared with iHA-24, iHA-100 dose-dependently decreased the number ofplaques and exhibited an anti-viral effect comparable to that ofzanamivir (Relenza, trade name)

Example 3

[Comparison in Treatment Effect Between Cyclic Peptide iHA-100 andZanamivir]

By the procedure shown in FIG. 5, iHA-100 cyclic peptide and zanamivirwere each administered to mice and their lethal rate due to viralinfection was studied.

The results are shown in FIGS. 6 and 7. Even in a group (iHA-100 6-10dpi) in which administration is started 6 days after infection, adecrease in lethal rate is observed. On the other hand, in azanamivir-administered group (FIG. 7), a dead individual is found evenwhen administration is started two days after infection and no treatmenteffect is obtained when administration is started fourth or more daysafter infection (FIG. 7).

Example 4

[Chemical Synthesis of Cyclic Peptide Binding to Hemagglutinin [2]]

In a manner similar to that of Example 1, the following peptides weresynthesized by modifying the peptide iHA-100 (SEQ ID NO: 6).

iHA-100-D4K (SEQ ID NO: 16)^(ClAc-)Trp-Thr-MeGly-Lys-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Arg-Cys-amide  iHA-100-S7A (SEQ ID NO: 17)^(ClAc-)Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeSer-His-Tyr-Thr-Val-Pro-Arg-Cys-amide iHA-100-S7K (SEQ ID NO: 18)^(ClAc-)Trp-Thr-MeGly-Asp-MePhe-MePhe-Lys-MeSer-His-Tyr-Thr-Val-Pro-Arg-Cys-amide  iHA-100-S7E (SEQ ID NO: 19)^(ClAc-)Trp-Thr-MeGly-Asp-MePhe-MePhe-Glu-MeSer-His-Tyr-Thr-Val-Pro-Arg-Cys-amide  iHA-100-P13hyP (SEQ ID NO: 20)^(ClAc-)Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Hyp-Arg-Cys-amide iHA-100-R14A (SEQ ID NO: 21)^(ClAc-)Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Ala-Cys-amide iHA-100-R14E (SEQ ID NO: 22)^(ClAc-)Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Glu-Cys-amide iHA-100-R14K (SEQ ID NO: 23)^(ClAc-)Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Lys-Cys-amide iHA-100-R14Dap (SEQ ID NO: 24)^(ClAc-)Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Dap-Cys-amide  iHA-100-COOH (SEQ ID NO: 25)^(ClAc-)Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Arg-Cys-COOH

In the above peptides, Dap represents 2,3-diaminopropionic acid and Hyprepresents 4-hydroxyproline.

Further, the following peptides were synthesized by cyclizing the abovepeptides, respectively.

iHA-100-D4K (SEQ ID NO: 26)Cyclo(Ac-Trp-Thr-MeGly-Lys-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Arg-Cys)-amide  iHA-100-S7A (SEQ ID NO: 27)Cyclo(Ac-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeSer-His-Tyr-Thr-Val-Pro-Arg-Cys)-amide  iHA-100-S7K (SEQ ID NO: 28)Cyclo(Ac-Trp-Thr-MeGly-Asp-MePhe-MePhe-Lys-MeSer-His-Tyr-Thr-Val-Pro-Arg-Cys)-amide  iHA-100-S7E (SEQ ID NO: 29)Cyclo(Ac-Trp-Thr-MeGly-Asp-MePhe-MePhe-Glu-MeSer-His-Tyr-Thr-Val-Pro-Arg-Cys)-amide  iHA-100-P13hyP (SEQ ID NO: 30)Cyclo(Ac-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Hyp-Arg-Cys)-amide  iHA-100-R14A (SEQ ID NO: 31)Cyclo(Ac-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Ala-Cys)-amide  iHA-100-R14E (SEQ ID NO: 32)Cyclo(Ac-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Glu-Cys)-amide  iHA-100-R14K (SEQ ID NO: 33)Cyclo(Ac-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Lys-Cys)-amide  iHA-100-R14Dap (SEQ ID NO: 34)Cyclo(Ac-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Dap-Cys)-amide  iHA-100-COOH (SEQ ID NO: 35)Cyclo(Ac-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr-Val-Pro-Arg-Cys)-COOH

Example 6

[Evaluation of Peptide Activity Against Influenza Virus [2]]

The growth inhibition activity of each of the cyclic peptidessynthesized in Example 4, that is, iHA-100, iHA-100-D4K, iHA-100-S7A,iHA-100-S7K, iHA-100-S7E, iHA-100-R14A, iHA-100-R14E, iHA-100-R14K,iHA-100-R14Dap, and iHA-100-COOH against influenza virus was evaluatedusing the plaque assay. Test was performed while setting contents ofeach peptide at 0.03 μM, 0.01 μM, 0.003 μM, 0.001 μM, 0.0003 μM, and0.0001 μM, respectively.

The results are shown in FIG. 9. All of the peptides showed adose-dependent decrease in the number of plaques. The peptidesiHA-100-R14A, iHA-100-R14E, and iHA-100-R14Dap showed a high anti-viraleffect. In particular, iHA-100-R14A and iHA-100-R14Dap showed a highanti-viral effect even when their concentration is low.

What is claimed is:
 1. A peptide comprising an amino acid sequence having 90% or more sequence identity to SEQ ID NO:
 1. 2. The peptide according to claim 1, wherein the peptide has an amino acid deletion, addition, or substitution at position 3, 6, 12, or 13 of SEQ ID NO:
 1. 3. The peptide according to claim 1, wherein the peptide is (SEQ ID NO: 1) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr- Val-Pro-Arg (SEQ ID NO: 36) Thr-MeGly-Lys-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr- Val-Pro-Arg; (SEQ ID NO: 37) Thr-MeGly-Asp-MePhe-MePhe-Ala-MeSer-His-Tyr-Thr- Val-Pro-Arg; (SEQ ID NO: 38) Thr-MeGly-Asp-MePhe-MePhe-Lys-MeSer-His-Tyr-Thr- Val-Pro-Arg; (SEQ ID NO: 39) Thr-MeGly-Asp-MePhe-MePhe-Glu-MeSer-His-Tyr-Thr- Val-Pro-Arg; (SEQ ID NO: 40) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr- Val-Hyp-Arg; (SEQ ID NO: 41) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr- Val-Pro-Ala; (SEQ ID NO: 42) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr- Val-Pro-Glu; (SEQ ID NO: 43) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr- Val-Pro-Lys; or (SEQ ID NO: 44) Thr-MeGly-Asp-MePhe-MePhe-Ser-MeSer-His-Tyr-Thr- Val-Pro-Dap.


4. The peptide according to claim 1, wherein said peptide is cyclized.
 5. The peptide according to claim 4, wherein said peptide comprises a chloroacetylated amino acid within 3 amino acids from the N-terminus; and cysteine within 3 amino acids from the C-terminus.
 6. The peptide according to claim 4, wherein said peptide comprises chloroacetyl-Trp at the N-terminus, having Cys at the C-terminus, and is cyclized via a thioether bond therebetween.
 7. A pharmaceutical composition comprising the peptide according to claim
 1. 8. The peptide according to claim 3, wherein said peptide is cyclized.
 9. The peptide according to claim 8, wherein said peptide comprises a chloroacetylated amino acid within 3 amino acids from the N-terminus; and cysteine within 3 amino acids from the C-terminus.
 10. The peptide according to claim 8, wherein said peptide comprises chloroacetyl-Trp at the N-terminus, having Cys at the C-terminus, and is cyclized via a thioether bond therebetween.
 11. A pharmaceutical composition comprising the peptide according to claim
 3. 